Microb Ecol DOI 10.1007/s00248-015-0583-x

ENVIRONMENTAL MICROBIOLOGY

Antibiotics, Antibiotic Resistance Genes, and Bacterial Community Composition in Fresh Water Aquaculture Environment in China Wenguang Xiong & Yongxue Sun & Tong Zhang & Xueyao Ding & Yafei Li & Mianzhi Wang & Zhenling Zeng

Received: 12 November 2014 / Accepted: 12 February 2015 # Springer Science+Business Media New York 2015

Abstract Environmental antibiotic resistance has drawn increasing attention due to its great threat to human health. In this study, we investigated concentrations of antibiotics (tetracyclines, sulfonamides and (fluoro)quinolones) and abundances of antibiotic resistance genes (ARGs), including tetracycline resistance genes, sulfonamide resistance genes, and plasmid-mediated quinolone resistance genes, and analyzed bacterial community composition in aquaculture environment in Guangdong, China. The concentrations of sulfametoxydiazine, sulfamethazine, sulfamethoxazole, oxytetracycline, chlorotetracycline, doxycycline, ciprofloxacin, norfloxacin, and enrofloxacin were as high as 446 μg kg−1 and 98.6 ng L−1 in sediment and water samples, respectively. The relative abundances (ARG copies/16S ribosomal RNA (rRNA) gene copies) of ARGs (sul1, sul2, sul3, tetM, tetO, tetW, tetS, tetQ, tetX, tetB/P, qepA, oqxA, oqxB, aac(6′)-Ib, and qnrS) were as high as 2.8×10−2. The dominant phyla were Proteobacteria, Bacteroidetes, and Firmicutes in sediment samples and Proteobacteria, Actinobacteria and Bacteroidetes in water samples. The genera associated with pathogens were also observed, such as Acinetobacter, Arcobacter, and Clostridium. This study comprehensively investigated Wenguang Xiong and Yongxue Sun contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00248-015-0583-x) contains supplementary material, which is available to authorized users. W. Xiong : Y. Sun : X. Ding : Y. Li : M. Wang : Z. Zeng (*) National Laboratory of Safety Evaluation (Environmental Assessment) of Veterinary Drugs, College of Veterinary Medicine, South China Agricultural University, 483 Wushan Road, Guangzhou 510642, China e-mail: [email protected] T. Zhang Environmental Biotechnology Laboratory, Department of Civil Engineering, The University of Hong Kong, Hong Kong, SAR, China

antibiotics, ARGs, and bacterial community composition in aquaculture environment in China. The results indicated that fish ponds are reservoirs of ARGs and the presence of potential resistant and pathogen-associated taxonomic groups in fish ponds might imply the potential risk to human health. Keywords Antibiotic resistance genes . Antibiotics . Bacterial community composition . Pathogens . Fish ponds

Introduction Increasing attention has been paid to environmental spread of antibiotic resistance genes (ARGs) due to their potential implications for human health. Evidences suggested that the environment may contribute to clinical antibiotic resistance [1, 2]. ARGs in the environment could be acquired by human pathogens via horizontal gene transfer, leading to difficult treatment of infectious disease. Although antibiotic resistance is ancient and naturally occurring phenomenon widespread in the environment [3], it is accelerated by antibiotic use in medicine and veterinary. The spread of ARGs in the environment is promoted by anthropogenic activities, such as animal farm [4], waste/wastewater treatment [5], and aquaculture [6]. To our knowledge, ARGs have been investigated in several environmental compartments, such as farm lands [7], rivers [8], and fish ponds [9]. However, the occurrence and distribution of environmental ARGs are still poorly understood. Aquaculture ponds have been proved as reservoirs for antibiotic resistance [10, 11]. Researchers have paid increasing attention to ARGs in aquaculture environment due to the rapid development of global aquaculture industry. The global aquaculture industry is dominated primarily by a few Asian countries, and China accounts for about 71 % of the total global aquaculture production [12]. Animal manure used as fertilizer

W. Xiong et al.

of aquaculture ponds (mainly fish ponds) is a common practice in integrated aquaculture-agriculture system in China. The nutrients in animal waste are consumed by fish and also support for the growth of photosynthetic organisms [12]. Animal manure and urine are continuously discharged directly into fish ponds in animal farm-fish pond system, a traditional household-based integrated farming system in China. The practice leads to the spread of ARGs and antibiotic resistance bacteria [13, 14] and may also change the bacterial community composition in aquaculture environment since the manure often contains antimicrobials used as growth promoters for terrestrial animals including swine and chicken. Aquaculture could accelerate the occurrence of antibiotic resistance [15]. Human might acquire ARGs via ingestion of contaminated aquaculture food (for example, fish and shrimp) and water. The largest scale of aquaculture production in China and the practice of antibiotic use in aquaculture farms require scientific evaluation of their impacts on ARGs in the environment. Previously, a few studies have investigated the occurrence and abundance of sulfonamide resistance genes, tetracycline resistance genes, and resistance bacteria in aquaculture environment [9, 16]. To our knowledge, however, the abundance of plasmid-mediated quinolone resistance (PMQR) genes has not been investigated in aquaculture environment by using a culture-independent method; it is also that, to our knowledge, the data on bacterial community composition in aquaculture environment are still limited. Guangdong province has the largest percentage of fish farming in China due to its geological advantages. Therefore, investigation of ARGs and bacterial community composition in aquaculture is especially necessary in Guangdong province of China. The objective of this study was to investigate concentrations of antibiotics, occurrence, and abundance of different types of ARGs and bacterial community composition in aquaculture environment in Guangdong, China. A comprehensive study was conducted, by (1) detecting the concentrations of antibiotics (tetracyclines, sulfonamides, and (fluoro)quinolones), (2) quantifying the abundances of ARGs (tetracycline resistance genes, sulfonamide resistance genes, and PMQR genes) by a cultureindependent method, and (3) analyzing bacterial community composition by amplicon sequencing.

Materials and Methods Sample Sites and Sample Collection Four sample sites marked 1, 2, 3, and 4 were from four fish ponds in Guangdong, China. Organic wastes from swine and poultry production are applied to these fish ponds two to five times per week at approximately 700–900 kg/ha/annum for nutrient cycle. Although no antimicrobials are used for therapeutic and prophylactic purpose in these fish ponds, different

classes of antibiotics are used in swine and poultry production. These antibiotics are poured into fish ponds by manure application for nutrient cycle. Paired sediment (∼200 g) and water (1 L) samples were collected from four corners of each fish pond. To avoid sampling site fluctuation, samples from different corners of each pond were pooled and labeled, e.g., for pond 1, 1-s, and 1-w. Each sample was subsequently divided into two aliquots that were used for antibiotic and molecular analysis, respectively. Each aliquot was further divided into three subsamples for repeated analysis. All aliquots were stored at −20 °C until further analysis. Antibiotic Analysis Typical antibiotics including sulfametoxydiazine, sulfamethazine, sulfamethoxazole, oxytetracycline, chlorotetracycline, doxycycline, ciprofloxacin, norfloxacin, and enrofloxacin were measured by using ultra-performance liquid chromatographyelectrospray tandem mass spectrometry. Water samples were filtered through Whatman qualitative filter paper to remove suspended solids. Weighted 1-g sediment and 50 mL filtered water were freeze-dried and extracted by using the buffer including acetonitrile/phosphate (v/v, 1:1, pH 3.0). Solid-phase extraction was conducted on Strata strong anion exchanger cartridges and Oasis hydrophilic-liphophilic balance cartridges based on the previous method of Li et al. [17]. The eluate was evaporated by nitrogen stream and redissolved in 0.1 % formic acid mixed with 99.9 % methanol/water (v/v, 1:9) solution. Antibiotic measurement was performed on an Agilent 1200 liquid chromatograph with a Waters Quattro Micro triple quadrupole mass spectrometer. The recovery percentages based on matrix-matched calibration were in the range of 63–83 % in sediment samples and 67–88 % in water samples. The limit of detection (LOD) was in the range of 3.8–9.6 μg/kg for sediment and 8.5–19 ng/L for water. PCR and Quantitative PCR DNA was extracted from sediment and water samples by using Power Soil DNA Kit and Power Water DNA Kit (Mo Bio Laboratories, Inc., CA, USA) according to the manufacturer’s instructions, respectively. The concentration and quality of DNA were checked by spectrophotometer analysis and agarose gel electrophoresis. Conventional PCR was used to determine the presence of all ARGs including sul1, sul2, sul3, tetM, tetO, tetW, tetS, tetQ, tetX, tetB/P, qepA, oqxA, oqxB, aac(6′)-Ib, and qnrS. PCR products with the expected size were purified and ligated into plasmid vectors before being cloned. Positive plasmids were extracted, purified, and sequenced for standard curves of quantitative PCR (qPCR). qPCR was performed by using SYBR® Premix Ex Taq™ II (TaKaRa, Dalian, China) following the manufacturer’s protocols. Positive and negative controls were performed for each

Resistance Genes and Bacterial Community in Aquaculture

amplification was performed by using Plantinum Taq Kit (Sangon Biotech (Shanghai) Co., Ltd., China) following the manufacturer’s protocols. Emulsion PCR was conducted using Ion Personal Genome Machine (PGM) Template 200 Kit according to the manufacturer’s instructions. DNA was extracted from three subsamples for each sample. The PCR products from the three subsamples were pooled together in equimolar ratios for amplicon sequencing. Amplicon sequencing was conducted on Ion Torrent PGM system with Ion 316™ chip according to Ion Sequencing 200 kit following the manufacturer’s protocols. To minimize effects of random sequencing errors, we removed (i) sequences that did not match the barcode and PCR primer, (ii) sequences that had more than one uncertain nucleotide, and (iii) sequences that were less than 100 bp. Both the barcode and the PCR primer were trimmed from the high-quality reads. Besides, we used LUCY (a trimming program) to remove other low-quality reads (Q

Antibiotics, Antibiotic Resistance Genes, and Bacterial Community Composition in Fresh Water Aquaculture Environment in China.

Environmental antibiotic resistance has drawn increasing attention due to its great threat to human health. In this study, we investigated concentrati...
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